Targeting c-Myc with a novel Peptide Nuclear Delivery Device.

Biologics such as peptides and antibodies are a well-established class of therapeutics. However, their intracellular delivery remains problematic. In particular, methods to efficiently inhibit intra-nuclear targets are lacking. We previously described that Pseudomonas Exotoxin A reaches the nucleoplasm via the endosomes-to-nucleus trafficking pathway. Here, we show that a non-toxic truncated form of PE can be coupled to peptides and efficiently reach the nucleoplasm. It can be used as a Peptide Nuclear Delivery Device (PNDD) to deliver polypeptidic cargos as large as Glutathione- S-transferase (GST) to the nucleus. PNDD1 is a fusion of PNDD to the c-myc inhibitor peptide H1. PNDD1 is able to inhibit c-Myc dependent transcription at nanomolar concentration. In contrast, H1 fused to various cell-penetrating peptides are active only in the micromolar range. PNDD1 attenuates cell proliferation and induces cell death in various tumor cell lines. In particular, several patient-derived Diffuse Large B-Cell Lymphomas cell lines die after exposure to PNDD1, while normal B-cells survive. Altogether, our data indicate that PNDD is a powerful tool to bring active cargo to the nucleus and PNDD1 could be the basis of a new therapy against lymphoma.

The NAE Pathway: Autobahn to the Nucleus for Cell Surface Receptors.

Various growth factors and full-length cell surface receptors such as EGFR are translocated from the cell surface to the nucleoplasm, baffling cell biologists to the mechanisms and functions of this process. Elevated levels of nuclear EGFR correlate with poor prognosis in various cancers. In recent years, nuclear EGFR has been implicated in regulating gene transcription, cell proliferation and DNA damage repair. Different models have been proposed to explain how the receptors are transported into the nucleus. However, a clear consensus has yet to be reached. Recently, we described the nuclear envelope associated endosomes (NAE) pathway, which delivers EGFR from the cell surface to the nucleus. This pathway involves transport, docking and fusion of NAEs with the outer membrane of the nuclear envelope. EGFR is then presumed to be transported through the nuclear pore complex, extracted from membranes and solubilised. The SUN1/2 nuclear envelope proteins, Importin-beta, nuclear pore complex proteins and the Sec61 translocon have been implicated in the process. While this framework can explain the cell surface to nucleus traffic of EGFR and other cell surface receptors, it raises several questions that we consider in this review, together with implications for health and disease.

Nuclear envelope-associated endosomes deliver surface proteins to the nucleus.

Endocytosis directs molecular cargo along three main routes: recycling to the cell surface, transport to the Golgi apparatus or degradation in endolysosomes. Pseudomonas exotoxin A (PE) is a bacterial protein that typically traffics to the Golgi and then the endoplasmic reticulum before translocating to the cytosol. Here we show that a substantial fraction of internalized PE is also located in nuclear envelope-associated endosomes (NAE), which display limited mobility, exhibit a propensity to undergo fusion and readily discharge their contents into the nuclear envelope. Electron microscopy and protein trapping in the nucleus indicate that NAE mediate PE transfer into the nucleoplasm. RNAi screening further revealed that NAE-mediated transfer depends on the nuclear envelope proteins SUN1 and SUN2, as well as the Sec61 translocon complex. These data reveal a novel endosomal route from the cell surface to the nucleoplasm that facilitates the accumulation of extracellular and cell surface proteins in the nucleus.

Proteomic analysis of interleukin enhancer binding factor 3 (Ilf3) and nuclear factor 90 (NF90) interactome.

Interleukin enhancer binding factor 3 (Ilf3) and Nuclear Factor 90 (NF90) are two ubiquitous proteins generated by alternative splicing from the ILF3 gene that provides each protein with a long and identical N-terminal domain of 701 amino acids and a specific C-terminal domain of 210 and 15 amino acids, respectively. They exhibit a high polymorphism due to their posttranscriptional and posttranslational modifications. Ilf3 and NF90 functions remain unclear although they have been described as RNA binding proteins but have been implicated in a large scale of cellular phenomena depending on the nature of their interacting partners, the composition of their protein complexes and their subcellular localization. In order to better understand the functions of Ilf3 and NF90, we have investigated their protein partners by an affinity chromatography approach. In this report, we have identified six partners of Ilf3 and NF90 that interact with their double-stranded RNA binding motifs: hnRNP A/B, hnRNP A2/B1, hnRNP A3, hnRNP D, hnRNP Q and PSF. These hnRNP are known to be implicated in mRNA stabilization, transport and/or translation regulation whereas PSF is a splicing factor. Furthermore, Ilf3, NF90 and most of their identified partners have been shown to be present in large complexes. Altogether, these data suggest an implication of Ilf3 and NF90 in mRNA metabolism. This work allows to establish a link between Ilf3 and NF90 functions, as RNA binding proteins, and their interacting partners implicated in these functions.

L-Ilf3 and L-NF90 traffic to the nucleolus granular component: alternatively-spliced exon 3 encodes a nucleolar localization motif.

Ilf3 and NF90, two proteins containing double-stranded RNA-binding domains, are generated by alternative splicing and involved in several functions. Their heterogeneity results from posttranscriptional and posttranslational modifications. Alternative splicing of exon 3, coding for a 13 aa N-terminal motif, generates for each protein a long and short isoforms. Subcellular fractionation and localization of recombinant proteins showed that this motif acts as a nucleolar localization signal. Deletion and substitution mutants identified four arginines, essential for nucleolar targeting, and three histidines to stabilize the proteins within the nucleolus. The short isoforms are never found in the nucleoli, whereas the long isoforms are present in the nucleoplasm and the nucleoli. For Ilf3, only the posttranslationally-unmodified long isoform is nucleolar, suggesting that this nucleolar targeting is abrogated by posttranslational modifications. Confocal microscopy and FRAP experiments have shown that the long Ilf3 isoform localizes to the granular component of the nucleolus, and that L-Ilf3 and L-NF90 exchange rapidly between nucleoli. The presence of this 13 aminoacid motif, combined with posttranslational modifications, is responsible for the differences in Ilf3 and NF90 isoforms subcellular localizations. The protein polymorphism of Ilf3/NF90 and the various subcellular localizations of their isoforms may partially explain the various functions previously reported for these proteins.

Genome-wide RNAi screens identify genes required for Ricin and PE intoxications.

Protein toxins such as Ricin and Pseudomonas exotoxin (PE) pose major public health challenges. Both toxins depend on host cell machinery for internalization, retrograde trafficking from endosomes to the ER, and translocation to cytosol. Although both toxins follow a similar intracellular route, it is unknown how much they rely on the same genes. Here we conducted two genome-wide RNAi screens identifying genes required for intoxication and demonstrating that requirements are strikingly different between PE and Ricin, with only 13% overlap. Yet factors required by both toxins are present from the endosomes to the ER, and, at the morphological level, the toxins colocalize in multiple structures. Interestingly, Ricin, but not PE, depends on Golgi complex integrity and colocalizes significantly with a medial Golgi marker. Our data are consistent with two intertwined pathways converging and diverging at multiple points and reveal the complexity of retrograde membrane trafficking in mammalian cells.